Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.2.30 (PARP)
 13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.
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PMID:Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid. 197 45

We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. 263, 17255-17257). In this study we have determined the amino acid sequence at its ADP-ribosylation site. Purified substrate was [32P]ADP-ribosylated by C1 botulinum toxin and digested with trypsin. The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin. By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X. During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues. Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene. These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product. This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h. The ADP-ribosylation of this protein affected neither its GTPase- nor its [35S]guanosine 5'-O-thiotriphosphate-binding activity.
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PMID:Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. 249 16